Element load, conventional biomarkers and altered protein expression profiles were studied in Carcinus maenas crabs, to assess contamination of “Domingo Rubio” stream, an aquatic ecosystem that receives pyritic metals, industrial contaminants, and pesticides. Lower antioxidative activities – glucose-6-phosphate and 6-phosphogluconate dehydrogenases, catalase – were found in parallel to higher levels of damaged biomolecules – malondialdehyde, oxidized glutathione –, due to oxidative lesions promoted by contaminants, as the increased levels of essential – Zn, Cu, Co – and nonessential – Cr, Ni, Cd – elements. Utility of Proteomics to assess environmental quality was confirmed, especially after considering the six proteins identified by de novo sequencing through capLC-μESI-ITMS/MS and homology search on databases. They include tripartite motif-containing protein 11 and ATF7 transcription factor (upregulated), plus CBR-NHR-218 nuclear hormone receptor, two components of the ABC transporters and aldehyde dehydrogenase (downregulated). These proteins could be used as novel potential biomarkers of the deleterious effects of pollutants present in the area.

The induction of cytochrome P4501A-mediated activity (e.g. ethoxyresorufin-O-deethylation, EROD) has been used as a biomarker for monitoring fish exposure to AhR-receptor ligands such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and polychlorinated dibenzo-dioxins/furans (PCDD/Fs). In this study we found that hepatic EROD is induced in fish (“Nile tilapia”, Oreochromis niloticus and “acará”, Geophagus brasiliensis) from the Guandu River (7-17-fold) and Jacarepaguá Lake (7-fold), Rio de Janeiro, Brazil. Since both cichlid fish are consumed by the local population and the Guandu River is the main source of the drinking water supply for the greater Rio de Janeiro metropolitan area, pollution by cytochrome P4501A-inducing chemicals is a cause for concern and should be further investigated in sediments, water and biota. We additionally showed that EROD activity in the fish liver post-mitochondrial supernatant--simpler, cheaper and less time consuming to prepare than the microsomal fraction--is sufficiently sensitive for monitoring purposes. Increased EROD activity in the liver of cichlid fishes indicated that Guandu River, the source of drinking water supply for Rio de Janeiro is polluted by CYP1A-inducing chemicals.

Understanding of xenobiotic metabolism is necessary for risk assessment as well as toxicological research. In the present study, nanoLC/LTQ-Orbitrap mass based non-targeted metabolomics method coupled with ultra-performance liquid chromatography (UPLC)/triple quadrupole mass based targeted metabolomics method was carried out to investigate the stereoselective metabolism of benalaxyl in mouse hepatic microsomes. As a result, 7 metabolites of benalaxyl were identified, including 5 previously reported and 2 newly identified metabolites in present work. Hydroxylation, oxidation and esterolysis were major biotransformation reactions of benalaxyl in mouse hepatic microsomes. For stereoselective metabolism study, (−)-R-benalaxyl degraded much faster than its antipode with the t1/2 of 81.24 and 190.38 min for (−)-R- and (+)-S-benalaxyl, respectively. More importantly, stereoselectivity was also observed in the formation of the identified metabolites. In conclusion, the combined use of the mass spectrometry based targeted and non-targeted metabolomics provided a new approach to investigate stereoselective metabolism and identify novel metabolites of chiral pesticides. This study highlights the stereoselective metabolic profile of benalaxyl enantiomers and provides reliable data for benalaxyl toxicological risk assessment in mammal.

Mercury is a recognized harmful pollutant in aquatic systems but still little is known about its sub-cellular partitioning in wild fish. Mercury concentrations in liver homogenate (whole organ load) and in six sub-cellular compartments were determined in wild Liza aurata from two areas – contaminated (LAR) and reference. Water and sediment contamination was also assessed. Fish from LAR displayed higher total mercury (tHg) organ load as well as in sub-cellular compartments than those from the reference area, reflecting environmental differences. However, spatial differences in percentage of tHg were only observed for mitochondria (Mit) and lysosomes plus microsomes (Lys+Mic). At LAR, Lys+Mic exhibited higher levels of tHg than the other fractions. Interestingly, tHg in Mit, granules (Gran) and heat-denaturable proteins was linearly correlated with the whole organ. Low tHg concentrations in heat stable proteins and Gran suggests that accumulated levels might be below the physiological threshold to activate those detoxification fractions.

Cytochrome P450 1A (CYP1A) is the major phase I of metabolic enzyme that plays essential roles in the detoxification of drugs and biotransformation of environmental pollutants. This study investigated CYP1A enzyme induction using EROD activity, CYP1A protein levels, and immunohistochemistry, along with histopathology of the liver, gills, kidneys, and intestine from the black rockfish, Sebastes schlegelii, exposed to benzo[a]pyrene (B[a]P). S. schlegelii has high risks of ingestion of sediment and absorption of heavy crude oil after accidental oil spills in Korea. This study thus exposed fish to B[a]P at 2, 20, and 200 μg/g body weight. EROD activity and CYP1A protein levels in hepatic microsomes had a positive correlation with the concentration of B[a]P (2–200 μg/g); in particular, exposure to 200 μg/g of B[a]P resulted in a 4- and 6-fold increase in hepatic EROD activity and CYP1A protein level, respectively. Hyperplasia of primary lamellar epithelium and atrophy of renal tubules were observed in the gills and kidney, respectively, following exposure to B[a]P at 200 μg/g. In contrast, severe histological alteration was not seen in intestinal tissues. Immunohistochemical analysis of the distribution of cellular CYP1A in four tissues showed strong immunostaining in the cytoplasm and nuclear membranes of the liver against B[a]P at 200 μg/g. Polycyclic aromatic hydrocarbons (PAHs), such as B[a]P, cause adverse histological changes in tissues of fish and provide evidence that PAH metabolism is inducible in fish liver, leading to increased CYP1A induction. Furthermore, the CYP1A induction in specific tissues might assist in monitoring and field assessment of marine ecosystems.

Although the debromination of polybrominated diphenyl ethers (PBDEs) in fish species has been studied, environmental factors, such as chemical contamination and habitat temperature, have not been well understood. This study compared debromination of BDE209 by hepatic microsomes of wild and cultured fish. PBDE concentrations in muscle tissue were lower in cultured fish than in wild fish. Debromination activity was high in wild common carp, followed by cultured common carp, moderate in cultured ayu sweetfish, and low in two cultured fish (rainbow trout and cherry salmon) and wild Japanese sea bass. Although common carps have been known as the species which have higher debromination ability, there were differences between wild and cultured common carps. First, wild common carp debrominated much more BDE209 than cultured common carp. Second was debromination of BDE209 lasted 96 h in wild carp but only 24 h in cultured carp. Wild carp were collected from warm wastewater effluent with consistently high concentrations of micropollutants. Cultured carp were collected from colder clean waters. Therefore, environmental factors in debromination include contamination or ambient temperature. To investigate the effects of habitat environment on debromination of PBDEs, we collected wild carp in summer and winter at two different locations with similar PBDE contamination levels. Carp collected from the natural river in winter had the highest BDE99 debromination activity. Although the results indicated seasonal difference of debromination of BDE209, we could not confirm whether habitat temperature or physiological cycle of carp affected to debromination ability. Thus, further investigation such as in vivo experiment is required.